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Recombinant\n                      <jats:italic toggle=\"yes\">E. coli<\/jats:italic>\n                      expressing wild-type LRA-5 and its engineered variants LRA-5\n                      <jats:sup>Y69Q<\/jats:sup>\n                      and LRA-5\n                      <jats:sup>V166E<\/jats:sup>\n                      showed MIC values equivalent to control strains. However, LRA-5\n                      <jats:sup>Y69Q\/V166E<\/jats:sup>\n                      displayed MICs above the resistant breakpoint for some \u03b2-lactams. Kinetic parameters correlated with the MICs, showing that the catalytic efficiency of LRA-5\n                      <jats:sup>Y69Q\/V166E<\/jats:sup>\n                      was comparable to those from class A \u03b2-lactamases, such as CTX-M-15, PER-2, and KPC-2. LRA-5\n                      <jats:sup>Y69Q\/V166E<\/jats:sup>\n                      exhibited\n                      <jats:italic toggle=\"yes\">k<\/jats:italic>\n                      <jats:sub>cat<\/jats:sub>\n                      \/\n                      <jats:italic toggle=\"yes\">K<\/jats:italic>\n                      <jats:sub>m<\/jats:sub>\n                      values up to 11,000-fold higher compared to wild-type LRA-5, which is associated with the presence of Glu166. The X-ray crystallographic structure of wild-type LRA-5 (1.80 \u00c5; PDB 8EO5) shows that the lack of both Glu166 and a deacylation water molecule contributes to a biologically insignificant activity. Interactions observed between LRA-5 and ceftazidime (2.35 \u00c5; PDB 8EO6) show structural conservation with other \u03b2-lactamases. In contrast, the crystallographic structure of LRA-5\n                      <jats:sup>Y69Q\/V166E<\/jats:sup>\n                      (2.15 \u00c5; PDB 8EO7) bears a deacylation water molecule that is associated with the increase in catalytic activity compared to the wild-type variant. Circular dichroism results confirm that amino acid substitutions in LRA-5 do not affect the overall content of the secondary\/tertiary structures. Evidence suggests that alternative evolutionary paths could have occurred for \u03b2-lactamases like LRA-5, produced by environmental microorganisms: (i) proteins having similar structural features than active \u03b2-lactamases may accumulate a small number of mutations (e.g., Y69Q\/V166E) to yield active enzymes and (ii) the \u03b2-lactamase fold may have lost key residues in the absence of antibiotics.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/aac.00675-25","type":"journal-article","created":{"date-parts":[[2025,11,28]],"date-time":"2025-11-28T12:49:32Z","timestamp":1764334172000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Proposal of metagenomic-origin LRA-5 as a precursor of active \u03b2-lactamases through Tyr69Gln and Val166Glu amino acid substitutions: a functional and structural 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